Protein isolation

ABSTRACT

A general method and strains of bacteria are described by means where the endogenous DNAK protein or homolog of the DNAK protein is tagged with a recognizable amino acid sequence and that through this tag, DNAK may be efficiently removed, and as such, recombinant protein purification may be greatly improved both in yield and purity with simplified purification steps that remove the DNAK and reduced cost, waste accumulation and labor, and the isolated recombinant protein will significantly benefit research and therapeutics in its application.

This application is a divisional of U.S. patent application Ser. No.14/497,314 filed Sep. 25, 2014, which claims benefit of U.S. provisionalapplication No. 61/882,569, filed on Sep. 25, 2013, in which areincorporated by reference herein their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for isolation of recombinantproteins, and namely methods that involve the removal of DNAK during theisolation procedure.

2. General Background and State of the Art

Obtaining substantial amounts of pure protein is essential ininnumerable biological studies and indispensable to the biochemicalcharacterization of proteins. The ease of growth, well-characterizedgenetics, and the large number of tools for gene expression has longmade Escherichia coli (E. coli) the organism of choice for proteinoverproduction.

DNAK is an abundant protein (about 1% of the total protein of E. coli)that interacts with a wide range of newly synthesized polypeptideswhereby it acts as a chaperone. Chaperone proteins assist in the properfolding of newly expressed proteins and assembly into oligomers and thusprevent protein aggregation through interaction. DNAK binding to otherproteins occurs when DNAK is bound to ADP, and release these proteinswhen bound to ATP. DNAK is also a required factor to disaggregatepreformed protein aggregates, and it participates in the degradation ofdamaged proteins through protease specific channels.

While important for protein production, DNAK contamination presents asignificant problem in protein purification. DNAK is able to bind tomany proteins that are not endogenous to the bacterial host strain.Moreover, DNAK contamination may prevent the separation of recombinantfusion protein production, impair analysis of unfolding-refoldingexperiments, and cause strong antigenic responses in rats and rabbitseven when the chaperone is present in trace amounts, which therebyaffects antibody production.

Some recombinant protein isolation methods utilize E. coli DNAK deletionstrains in order to eliminate DNAK contamination, but only when DNAK isnot required to improve the solubility and the quality of the isolatedrecombinant proteins. Still, these deletions strains have narrowerranges of permissive temperatures for growth and exhibit multiplecellular defects which may reduce the overall yield of recombinantprotein as well as stability of the strain.

Other methods involving fusion protein production utilize software basedalgorithms that determine appropriate amino acids surrounding theputative DNAK binding site of the recombinant protein and alter thesequence in order to decrease putative affinity for DNAK. Uponpurification of these fusion protein bound to resin, MgATP plus solubledenatured E. coli proteins are used to wash the protein prior toelution. However, such methods are effective in only a limited number ofcases, and not generally for all fusion proteins. Also, they do noteliminate contamination of their isolated recombination proteins thatinherently contain putative DNAK binding sites. In addition, reagents ofthis procedure agents are costly and some are not suitable forcommercial or therapeutic use (e.g. glycerol).

Alternate methods employ co-chaperone proteins which bind to DNAK in anyof the nucleotide bound states. The co-chaperone protein was histidinetagged at the N-terminus, and this fusion recombinant protein wasisolated by a one-step purification with nickel affinity chromatography.Despite the ability to remove DNAK via this co-chaperone, the methoddoes not completely eliminate the DNAK, and relies on the transformationof an additional recombinant protein which reduces cellular resourcesneeded to express the target recombinant protein at higher levels.

INVENTION SUMMARY

The invention is a method to remove DNAK during a recombinant proteinpurification. The method requires the use of a bacterial strain,commonly used for recombinant protein production, which is able toexpress a tagged DNAK. The bacterial strain may be E. coli. The proteintag may be incorporated into the genomic copy of the DNAK gene,resulting in a strain which produces the tagged DNAK endogenously. Thestrain may be a mutant strain wherein the endogenous DNAK has beendeleted and the tagged DNAK is expressed from an introduced vector. Thetag may be added to DNAK or a homolog of DNAK with the similar functionas DNAK. The tag may be a histidine tag, myc tag or any equivalent tagswherein the tag is effective in the removal of the tagged protein whenused with a resin based solid or liquid phase method or any otherpurification techniques that employ the use of tag for proteinisolation.

The presence of DNAK is important for optimal replication of thebacterial culture which in turn allows for greater expression and yieldof the target recombinant protein. The nucleotide sequence encoding theprotein tag may be inserted into any region of the DNAK sequence thatdoes not affect the function of DNAK and therefore, does not impairbacterial growth.

The tagged DNAK bacterial strain will first be transformed with thevector of the target recombinant protein. After the transformedbacterial culture has been incubated to the optimal level of growth, thecellular extract may be collected by standard means of isolation. Thecellular extract may then be applied to resin containing the tag'sligand directed toward the tagged DNAK. When used in chromatography, theDNAK and any DNAK-bound protein remains bound to the resin, and theeluate contains the target recombinant protein. Further isolation of thetarget recombinant proteins maybe employed. In alternate methods, taggedDNAK may be removed after the target recombinant protein has beeninitially isolated. Other methods may include subsequent rounds oftagged DNAK isolation.

The novel features which are characteristic of the invention, both as tostructure and method of operation thereof, together with further objectsand advantages thereof, will be understood from the followingdescription, considered in connection with the accompanying drawings, inwhich the preferred embodiment of the invention is illustrated by way ofexample. It is to be expressly understood, however, that the drawingsare for the purpose of illustration and description only, and they arenot intended as a definition of the limits of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1. The exemplary gels that show the efficacy of DNAK removal.

FIG. 2. The mass spectroscopy data obtained for Band 1 as shown in thegel for the identification of the DnaK isolated band.

FIG. 3. The MASCOT data of Band 1 shown in FIG. 2 wherein SEQ ID No. 12is the amino acid sequence of DNAK protein, SEQ ID No. 13 is the aminoacid sequence of DNAK protein with a double strep tag fused to theC-terminus, and SEQ ID Nos. 14-45 are the MASCOT peptide fragments ofDNAK protein.

FIG. 4. The mass spectroscopy data used on the Band 2 as shown in thegel for the identification of the GRPE isolated band.

FIG. 5. The MASCOT data of Band 2 shown in FIG. 4 wherein SEQ ID No. 46is the amino acid sequence of GRPE protein, SEQ ID No. 47 is the aminoacid sequence of GRPE protein suggested by NCBInr, and SEQ ID Nos. 48-59are the MASCOT peptide fragments of GRPE protein.

FIG. 6. The growth curve of wild type BL21 strain.

FIG. 7. The growth curve of the BL21 DNAK STREP strain.

DETAILED DESCRIPTION OF THE INVENTION (i) Definitions

The following definitions, unless otherwise stated, apply to all aspectsand embodiments of the present application.

The term “DNAK” refers to an E. coli protein that plays an essentialrole in the initiation of phage lambda DNA replication, where it acts inan ATP-dependent fashion with the DnaJ protein to release lambda O and Pproteins from the preprimosomal complex. DnaK is also involved inchromosomal DNA replication, possibly through an analogous interactionwith the DnaA protein. Also the protein participates actively in theresponse to hyperosmotic shock.

The term “GrpE” refers to an E. coli protein that participates activelyin the response to hyperosmotic and heat shock by preventing theaggregation of stress-denatured proteins, in association with DnaK andGrpE. It is the nucleotide exchange factor for DnaK and may function asa thermosensor. Unfolded proteins bind initially to DnaJ; uponinteraction with the DnaJ-bound protein, DnaK hydrolyzes its bound ATP,resulting in the formation of a stable complex. GrpE releases ADP fromDnaK; ATP binding to DnaK triggers the release of the substrate protein,thus completing the reaction cycle. Several rounds of ATP-dependentinteractions between DnaJ, DnaK and GrpE are required for fullyefficient folding.

A “plasmid” is a vector that refers to an independently replicatingcircular double-stranded piece of DNA. The plasmid may contain an originof replication such as the E. coli oriC, an selectable antibioticresistance gene conferring resistance to but not limited to β-lactam,macrolide, and aminoglycosides antibiotics, a promoter sequence underexpression control, and a multiple cloning site containing restrictionsites.

The plasmid may be an “expression plasmid”. Expression plasmids allowfor the expression of a cloned gene. An expression plasmid contains aninducible promoter region that allows for the regulation and inductionof gene expression of a gene cloned into the plasmid's multiple cloningsite, a ribosomal binding site, a start codon, a stop codon, and atermination of transcription sequence.

The term “promoter sequence” is a region of DNA either upstream ordownstream from the site of initiation of transcription of a gene. Asused herein, a bacterial promoter includes necessary consensus sequencesof TTGACA at the −35 and a Pribnow box TATAAT sequence at the −10position upstream of the start of transcription, and may also contain anUP element upstream of the −35 region.

“BL21-(DE3) is an E. coli strain that is chemically competent fortransformation and protein expression. The strain may express proteinsunder the control the T7 promoter. The strain is deficient in proteaseLon and OmpT.

The term “transformation” refers to a process of introducing exogenousgenetic material into a bacterium by methods employing membranepermeability via chemical or electrical means. Performing atransformation involves adding genetic material, such as a plasmid, toan aliquot of competent bacterial cells, such as E. coli, and allowingthe mixture to incubate on ice. The bacterial cells are then eitherelectroporated or placed at 42° C. for approximately 1 minute and thenreturned to incubate on ice. The bacterial cells are then grown on anagar plate overnight until colonies are visible. The agar plate maycontain antibiotic or nutrient conditions for colony selection.

The term “transfection” refers is the process of deliberatelyintroducing nucleic acids into cells. The term is often used fornon-viral methods in eukaryotic cells. It may also refer to othermethods and cell types, although other terms are preferred:“transformation” is more often used to describe non-viral DNA transferin bacteria, non-animal eukaryotic cells, including plant cells. Inanimal cells, transfection is the preferred term as transformation isalso used to refer to progression to a cancerous state (carcinogenesis)in these cells. “Transduction” is often used to describe virus-mediatedDNA transfer. Nature Methods 2, 875-883 (2005)

The term “homologous sequence” refers to an amino acid or nucleotidesequence that is at least 70% to 99% homologous to a correspondingreference sequence. Sequences that are 90% identical have no more thanone different amino acid per 10 amino acids in the reference sequence.The percentage of homology between two or more sequences may beidentified using a homology algorithm of Smith and Waterman (1970) Adv.Appl. Math 2:482c, Needleman and Wunsch (1970) J. Mol. Biol. 48:433, orPearson and Lipman (1988) Proc. Natl. Sci. 85:2444. The methods ofsequence alignment are known to those in the art. A computer basedprogram employing the mentioned or alternative sequence comparisonalgorithms may be used such as BLAST as described in The NCBI Handbook(2002) or ClustalOmega as described in Sievers et. al. Mol. Sys. Bio.7:539 (2011).

“Homologous recombination” refers to a type of genetic recombination inwhich nucleotide sequences are exchanged between two similar oridentical molecules of DNA. It is most widely used by cells toaccurately repair harmful breaks that occur on both strands of DNA,known as double-strand breaks. Although homologous recombination varieswidely among different organisms and cell types, most forms involve thesame basic steps. After a double-strand break occurs, sections of DNAaround the 5′ ends of the break are cut away in a process calledresection. In the strand invasion step that follows, an overhanging 3′end of the broken DNA molecule then “invades” a similar or identical DNAmolecule that is not broken. After strand invasion, the further sequenceof events may follow either of two main pathways discussed below (seeModels); the DSBR (double-strand break repair) pathway or the SDSA(synthesis-dependent strand annealing) pathway. Homologous recombinationis conserved across all three domains of life as well as viruses,suggesting that it is a nearly universal biological mechanism. Alberts,B et al (2002). “Chapter 5: DNA Replication, Repair, and Recombination”

“Recombinant” DNA or protein is used to refer to DNA molecules formed bylaboratory methods of genetic recombination (such as molecular cloning)to bring together genetic material from multiple sources, creatingsequences that would not otherwise be found in biological organisms orused to refer to the proteins that are encoded by the recombinant DNA.Recombinant DNA are sometimes called chimeric DNA.

“Restriction Endonucleases” refer to enzymes that cut DNA at or nearspecific recognition nucleotide sequences known as restriction sites.Roberts R J (November 1976). “Restriction endonucleases”. CRC Crit. Rev.Biochem. 4 (2): 123-64.

The term “amplification” refers to the act of mass replication of agenetic sequence. Amplification of a genetic sequence may be performedby PCR using primers that hybridize to flanking ends of a geneticsequence of interest. Amplification of a genetic sequence may also beperformed in vivo by transforming bacteria with a plasmid ortransfecting a host cell with a virus that carries the recombinantgenetic sequence of interest.

The term “protein expression” refers to the production of protein withina host cell such as a bacteria, yeast, plant, or animal cell. A vectorcarrying the coding sequence for a recombinant protein under the controlof a promoter, such as an expression plasmid, is inserted into a hostcell. The promoter controlling the expression of the recombinant gene isthen induced and the protein encoded by the recombinant gene is producedwithin the host cell.

The term “protein coding sequence” refers to a portion of a gene thatcodes for a polypeptide. The coding sequence is located between an ATGinitiation of translation codon and the location of a TAG, TAA, or TGAtermination of translation codon. Typical to eukaryotic genes, thecoding sequence may include the “exons” of a gene, which is the sequenceof a gene that is transcribed and translated into a polypeptide, and mayexclude the “introns” of a gene, which is the sequence of a gene that istranscribed but not translated into a polypeptide.

The term “protein purification” refers to a process of purifying aprotein and may employ any technique used to separate and isolate aprotein of interest to a satisfactory level of purity. Proteinpurification exploits a protein's various properties such as size,charge, binding affinity, and biological activity. Liquid columnchromatography is commonly used in protein purification where a celllysate containing an expressed protein is passed over a “resin” withparticular binding affinity for the protein of interest. A resin is acompound or a polymer with chemical properties that supports thepurification of proteins via ion exchange, hydrophobic interaction, sizeexclusion, reverse phase, or affinity tag chromatography. A protein mayalso be purified by non-chromatographic techniques such as through theelectroporation of protein from an excised piece of a polyacrylamide gelthat contained a protein sample of interest.

The term “MALDI” refers to matrix-assisted laser desorption ionizationwhich is a mass spectrometry technique used to analyze compounds, andbiomolecules such as polypeptides and proteins, by determining theirmolecular masses. A protein sample is first prepared for MALDI byenzymatic digestion with a protease such as trypsin. The sample is thenchemically coupled to a matrix and then introduced into the massspectrometer. A pulsed laser beam targets the sample which results indesorption and ionization of the polypeptide from a solid to a gasphase. The vaporized ions are accelerated in an electric field towards adetector. Peptide fragments are then identified based on theirmass-to-charge ratio via peptide mass fingerprinting or tandem massspectrometry. The peptide masses are displayed as a list of molecularweight peaks which are then compared to a database of known peptidemasses such as that of Swissprot allowing for a statisticalidentification of the original protein sample.

As used here, the term “MASCOT” refers to algorithm called MASCOT(Matrix Sciences) which is a search engine that is used for theidentification and characterization of mass spectrometry protein data.The probalistic scoring depends on which of the matched proteins has thelowest probability of occurring by chance and is thus returned with themost significant match. A protein score of 85 or above (whichcorresponds to a p<0.05) is considered to be significant.

A “protein tag” refers to an amino acid sequence within a recombinantprotein that provides new characteristics to the recombinant proteinthat assist in protein purification, identification, or activity basedon the tag's characteristics and affinity. A protein tag may provide anovel enzymatic property to the recombinant protein such as a biotintag, or a tag may provide a means of protein identification such as withfluorescence tags encoding for green fluorescent protein or redfluorescent protein. Protein tags may be added onto the N- or C-terminusof a protein. A common protein tag used in protein purification is apoly-His tag where a series of approximately six histidine amino acidresidues are added which enables the protein to bind to proteinpurification matrices chelated to metal ions such as nickel or cobalt.Other tags commonly used in protein purification include Strep tag,chitin binding protein, maltose binding protein,glutathione-S-transferase, and FLAG-tag. Tags such as “epitope tags” mayalso confer the protein to have an affinity towards an antibody. Commonantibody epitope tags include the V5-tag, Myc-tag, and HA-tag.

The terms “fusion protein” or “fused protein” refer to a protein that iscoded by a single gene and the single gene is made up of codingsequences that originally coded for at least two or more separateproteins. A fusion protein may retain the functional domains of the twoor more separate proteins. Part of the coding sequence for a fusionprotein may code for an epitope tag. As described herein for theantibody like protein, a fusion protein may also contain sequences thatcode for a variety of proteins having vary functional roles based on itsapplication. “polypeptides”

“Chromatography” is the collective term for a set of laboratorytechniques for the separation of mixtures. The mixture is dissolved in afluid called the mobile phase, which carries it through a structureholding another material called the stationary phase. The stationaryphase may be referred to as a “resin”. The various constituents of themixture travel at different speeds, causing them to separate. Theseparation is based on differential partitioning between the mobile andstationary phases. Subtle differences in a compound's partitioncoefficient result in differential retention on the stationary phase andthus changing the separation.

Chromatography may be preparative or analytical. The purpose ofpreparative chromatography is to separate the components of a mixturefor more advanced use (and is thus a form of purification). Analyticalchromatography is done normally with smaller amounts of material and isfor measuring the relative proportions of analytes in a mixture. The twoare not mutually exclusive.

Size-exclusion chromatography or column (SEC) is also known as gelpermeation chromatography (GPC) or gel filtration chromatography andseparates molecules according to their size (or more accuratelyaccording to their hydrodynamic diameter or hydrodynamic volume).Smaller molecules are able to enter the pores of the media and,therefore, molecules are trapped and removed from the flow of the mobilephase. The average residence time in the pores depends upon theeffective size of the analyte molecules. However, molecules that arelarger than the average pore size of the packing are excluded and thussuffer essentially no retention; such species are the first to beeluted. It is generally a low-resolution chromatography technique andthus it is often reserved for the final, “polishing” step of apurification. It is also useful for determining the tertiary structureand quaternary structure of purified proteins, especially since it canbe carried out under native solution conditions.

“Affinity chromatography” is a method of separating biochemical mixturesbased on a highly specific interaction such as that between antigen andantibody, enzyme and substrate, or receptor and ligand. The term“column” may be used instead coupled with an explanation of the type ofresin that is bound

In molecular cloning, a “vector” is a DNA molecule used as a vehicle toartificially carry foreign genetic material into another cell, where itcan be replicated and/or expressed. A vector containing foreign DNA istermed recombinant DNA. The four major types of vectors are plasmids,viral vectors, cosmids, and artificial chromosomes. Common to allengineered vectors are an origin of replication, a multiple cloningsite, and a selectable marker.

The term flippase recognition target “FRT” refers to the DNA sequencethat is recognized by the enzyme called flippase which is responsible inpart for homologous recombination. The 34 bp minimal FRT site sequencehas the sequence 5′-GAAGTTCCTATTCtctagaaaGtATAGGAACTTC-3′ (SEQ ID No.60) for which flippase (Flp) binds to both 13-bp 5′-GAAGTTCCTATTC-3′(SEQ ID No. 61) arms flanking the 8 bp spacer, i.e. the site-specificrecombination (region of crossover) in reverse orientation. FRT-mediatedcleavage occurs just ahead from the asymmetric 8 bp core region(5′tctagaaa3′) on the top strand and behind this sequence on the bottomstrand. Several variant FRT sites exist, but recombination can usuallyoccur only between two identical FRTs but generally not amongnon-identical (“heterospecific”) FRTs. Zhu X D, Sadowski P D (1995). ZhuX D, Sadowski P D (1995). “Many available constructs include anadditional arm sequences (5′-GAAGTTCCTATTCC-3′) (SEQ ID No. 62) one basepair away from the upstream element and in the same orientation:5′-GAAGTTCCTATTCcGAAGTTCCTATTCtctagaaaGtATAGGAACTTC-3′ (SEQ ID No. 63).This segment is dispensable for excision but essential for integration,including Recombinase-mediated cassette exchange”; Turan, S., Bode, J.(2011). “Site-specific recombinases: from tag-and-target- totag-and-exchange-based genomic modifications”. FASEB J. 25: 4088-4107.

“Cleavage-dependent Ligation by the FLP Recombinase”. Journal ofBiological Chemistry 270 (39): 23044-54 Schlake T, Bode J (1994). “Useof mutated FLP recognition target (FRT) sites for the exchange ofexpression cassettes at defined chromosomal loci”. Biochemistry 33 (43):12746-12751.

The term “contaminants” may include DNA, RNA, nucleotides, cofactors,cellular debris such as peptidoglycans, cell wall and/or cell wallcomponents, organelles, peptides and/or polypeptides, select proteins,or any other molecule or component which is undesirable following thepurification of a protein of interest.

(ii) Polypeptide Sequences and Agents of the Application

Precise alterations were made in the genome of E. coli using the methodof Link et al., 1997. Integrations are based on the pKO plasmids, pMH9and pTOF24 (Merlin et al. 2002). These plasmids have the followingfeatures: chloramphenicol resistance gene, temperature sensitive ORIwith functionality at 30° C., but not 37-42° C., and the sacB gene,which renders cells sensitive to the presence of sucrose in growthmedia. An exemplary embodiment of the sequence of DNAK inserted into thepKO plasmid is in SEQ ID. 1.

An 800 bp region of homology to the E. coli genome is cloned at therestriction endonuclease sites of PstI and SalI of the pKO plasmid. The800 bp region may be constructed using crossover PCR. The 800 bp DNAwill contain additional alterations such as the tag encoding nucleotidesequences. Any such alterations will have 400 bp of homology flanking oneither side of the location of the tag encoding nucleotide sequence.Alterations may also include adding unique restriction sites.

In an exemplary embodiment, the primers that may be used to amplify thetagged DNAK gene are depicted in SEQ ID. 2 & 3. It is understood by onewith the ordinary skill of the art that other primers and locations ofthe tags and sites of homologous recombination maybe used to amplify thetagged DNAK gene.

The amplified DNAK sequence modified with a tag was inserted between twoFRT cassettes present within an expression vector.

In the present application, one embodiment of the tagged DNAK genesequence may contain an amino acid Strep-tag sequence(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that was added to the C-terminus endedthrough DNA recombination as shown in SEQ ID. 9. A tag could also beadded to the N-terminus region. In alternate embodiments, other tagssuch as a Flag-Tag, His-tag or Myc-tag or the like may also be used.

In the exemplary embodiment, the recombinant plasmids were transformedinto recipient cells with chloramphenicol selection at 30° C. Recipientcells may be the BL21 (DE3) strain or the like. The DNAK strain was thenallowed to homologously recombine with the E. coli chromosome.

It is well known to those with ordinary skill in the art that othercells that contain homologs of DNAK could be genetically modified withthe homolog containing a recognizable tag based on this exemplaryembodiment. Primers may be constructed to amplify the DNAK homolog withthe primers having the nucleotide sequence that encodes for therecognizable tag.

Colonies were grown on LB agar plates with chloramphenicol overnight at42° C., and colonies were selected based on size. Selected colonies thatdemonstrated successful homologous recombination were then crossselected for growth on LB agar with 5% W/V sucrose at 37° C. Crossselected colonies were then simultaneously “patch tested” on LB agarcontaining 5% sucrose and on LB agar containing 5% sucrose and 5%chloramphenicol. Chloramphenicol sensitive colonies were screened by PCRand sequenced.

The modified DNAK or a homolog of a naturally occurring DNAK wasisolated from the strain containing the homologously recombined taggedDNAK encoding gene to confirm a protein sequence through the use of theadded tag. Chromatography or an equivalent method that targets the tagmay be used to isolate the resultant protein.

In the present embodiment, the bacterial lysate of the modified strain,BL21-DNAK-strep may be applied to a Strep-tactin resin column. The elutefractions may then be run on an SDS-protein gel as shown in FIG. 2. TheStrep-tagged DNAK is shown with a molecular weight that corresponds toits estimated 72.4 kDa molecular weight. An additional lower molecularweight band was also observed and subsequent analysis revealed that theidentity of the protein was Grp E, a commonly associated co-chaperoneprotein of DNAK. See the data below. (Sugimoto et al, Prot. Exp. AndPur., 60 31-36 (2008).

The bands may then be extracted from the gel and prepared forsequencing. The Strep-tagged DNAK may be partially digested and theresultant fragments isolated for the purposes of MALDI mass fingerprintanalysis. In the present invention, the modified protein was subjectedto a partial tryptic digestion. Digested fragments were then run througha Micromass MALDI microMX mass spectrometer. FIG. 2 shows the massspectroscopy data corresponding to FIG. 2 the gel of band 1 of themodified DNAK molecule. The data was then analyzed using the MASCOTalgorithm which matched the mass spectrometer data to the DNAK geneproduct (E. coli 0157:H7 str. EDL9331] with the highest protein score of412 as shown in FIG. 3.

Band 2 from the SDS-PAGE of FIG. 4 was also analyzed under MALDI and hada MASCOT protein score of 185 for the grpE gene product [E. coli 0157:H7str. EDL933]. The co-elution of GrpE with Strep-tagged DNAK demonstratesthat the modified DNAK has retained its structural and functionalconfirmation as it is capable of binding and co-eluting with GrpE.

Based on the MASCOT results, the partial protein sequence is depicted inFIG. 5.

With regards to studying bacterial strains of endogenous proteins, thepresent invention would not sacrifice bacterial viability and growth. Inthe exemplary modified strain, the resultant modified strain may then bemonitored to growth. In the exemplary embodiment the modified strain,BL21-DNAK Strep strain and the wild-type BL21 (DE3) strain wereseparately inoculated into LB media and growth was observed over 630minutes (10.5 hours) which is consistent with the mid-log phase growth.Mid-log phase growth is the optimal for growth phase when expressingrecombinant protein in bacteria. See FIGS. 6 & 7. Growth curvesdemonstrated that there was no substantial difference bacterial growthover this period.

Protein level expression remains unaffected by the presence of thetagged DNAK given that the functionality of DNAK is still able tofunctionally interact with GrpE as shown in FIG. 1. DNAK removal is alsofar superior than previous methods used to remove DNAK. In the exemplaryembodiment, a BL21-DNAK strain transformed with recombinant proteinvector that ultimately expresses a 30 kDa and 36 kDa monomer proteinsand the strain was grown to mid-log phase. The cell lysate was isolatedand applied to a size exclusion column (SEC) as shown in FIG. 1.A. Therecombinant protein found in fractions corresponding to the hydrodynamicmolecular weight range of the native recombinant protein were collectedand run on an SDS-PAGE with the earliest collected fraction in lane 1.The DNAK band was observed around 72 kDa.

SEC fractions from lane 2-8 were pooled and applied to a gravity flowStrep-tactin resin column. FIG. 2.B shows an aliquot of the lanes forthe input sample (lane 1) and the elute fractions (lanes 2-5) which wereeluted by 2.5 mM D-Desthiobiotn. The tagged DNAK protein was the onlyprotein to visibly remain with no detectable presence of the 30 kDa or36 kDa recombinant protein.

FIG. 1.C shows the pooled flow fraction from the Strep-tactin resincolumn used for FIG. 1.B which was reapplied to an SEC. Thecontaminating tagged DNAK was significantly removed with minimal loss ofthe recombinant protein. FIG. 1D shows that there are still tracesamounts of bound protein coupled with the tagged DNAK.

(iii) Uses

The present invention provides a crucial solution to the problem ofreducing DNAK contamination of another targeted isolated protein withoutaffecting the viability of the bacteria, optimal protein productionlevels, and the proper folding and formation of the expressedrecombinant protein. The present invention also does not require the useof reagents that are costly or prohibited for recombinant sample thatwould be used for commercial or therapeutic applications.

A recognizable tag may be inserted into any region of the DNAK sequencethat does not significantly impair the function of DNAK as well asbacterial growth. However, tags that do partially impair DNAK functionmay be used for recombinant proteins that have a weak affinity for DNAK.The tag may be a protein tag and may be, but not limited to a Strep-,Myc-, His-, or Flag tag. Multiple tags may be added to DNAK to enhancethe selective and isolation.

The tag may also be a recognizable ribonucleic acid tag whereinexpression of DNAK may be stopped at the RNA level by factors thatrecognize the tag and inhibit translation. Such a tag may provideanother means of inhibiting DNAK production during the varying growthphases of the bacteria.

While one of the exemplary embodiments of the present invention utilizea BL21 strain, any bacterial strain and its chromosomal DNAK gene may bemodified as provided in this present invention.

The present invention may be used to replace DNAK homologs or otherchaperone proteins present in eukaryotic cells such as yeast ormammalian cells or cell lines. Such application may improve commercialapplications for recombinant protein expression that require posttranslational modifications.

In the present invention, a tagged DNAK bacterial strain may betransformed with the vector of the target recombinant protein. After thetransformed bacterial culture has been incubated to the optimal level ofgrowth, the cellular extract may be collected by standard means ofisolation known to those with ordinary skill in the art. The cellularlysate may then be applied to isolation method that targets the tag ofthe tagged DNAK protein. Such methods include solid phase supportisolation such as chromatography or resins attached to tubes orequivalent solid supports. In the alternate liquid phase separation maybe employed. An example of a liquid phase separation may involve amagnetic bead that is attached to a ligand that may be bound by the tagthat is attached to the DNAK.

In alternate methods, tagged DNAK may be removed after the targetrecombinant protein has been initially isolated. Other methods mayinclude subsequent rounds of tagged DNAK isolation.

The use of this bacterial strain will also save cost on expensivepurification procedures. Recombinant protein expression and isolation isone of the most widely used methods in research and commercial industry.Reagents such as ATP and glycerol can be eliminated. In addition, havingendogenous DNAK tag strains reduces the need to transform bacterialstrains with a plasmid into DNAK deletion strains resulting in greaterconsistency in experimental results as well as a reduction in cost andtime.

The elimination for the need of a DNAK tag plasmid also reduces thechance that the recombinant proteins plasmid or the DNAK tag plasmid islost during bacterial growth.

DNAK tag bacterial strains may be used to produce recombinant proteinsfor therapeutic use where the chances of immune responses stemming fromDNAK or DNAK bound proteins other than the recombinant protein areeliminated or lowered. In addition, the use of the inventive strainreduces the need for use of reagents that may have a toxic effect ifpresent in medicinal preparation of the isolated recombinant protein.

Tagged DNAK may be also be used when a particular protein that is knownto bind to DNAK may be isolated when the particular protein is expressedand isolated from DNAK tag strains. This may be useful for bothdiagnostic and research applications.

Tagged DNAK amino acid sequence could also be modified with proteasesensitive recognition sites and such recognition sites may be employedto along with the tagged DNAK to further decrease any trace amounts ofDNAK.

While the specification describes particular embodiments of the presentinvention, those of ordinary skill can devise variations of the presentinvention without departing from the inventive concept.

What is claimed is:
 1. A method of protein isolation comprising: a)transfecting a first nucleic acid sequence that encodes for a firstprotein having a first tag, wherein the first tag's nucleic acidsequence does not encode for a 6-His tag, into a cell wherein thechromosome has been genetically modified to express an endogenous DNAKprotein or a DNAK protein homology with a 6-His tag wherein the firstprotein is not the endogenous DNAK protein or the DNAK protein homolog:b) expressing the first protein; c) extracting cell lysate from thecell; d) a series of purifying steps comprising, in no particular order,an endogenous DNAK protein or DNAK protein homolog purifying step forremoving the endogenous DNAK protein or DNAK protein homolog bytargeting the 6-His tag; and e) a purifying step for isolating the firstprotein through a method that binds to the first tag.
 2. The method ofclaim 1 wherein said cell is a bacterial cell.
 3. The method of claim 1wherein said first tag is an epitope tag.
 4. The method of claim 1wherein at least one of the purifying steps utilizes a solid phasesupport or liquid phase support isolation procedure.
 5. The method ofclaim 1 wherein at least one of the purifying step may be repeated atleast once.
 6. The method of claim 1 wherein additional purifying stepsare employed that target other contaminants within the cell lysate. 7.The method of claim 1 wherein the endogenous DNAK protein or DNAKprotein homolog purifying step also removes another protein that isbound to the endogenous DNAK protein or DNAK protein homolog.
 8. Amethod of protein isolation that comprising: a) transforming a nucleicacid that encodes for a first protein and a first tag into a bacterialcell wherein the chromosome has been genetically modified to express anendogenous DNAK protein or DNAK protein homolog and a second tag,wherein the first protein is not the endogenous DNAK protein or DNAKprotein homolog, and wherein the first tag and second tag are not thesame; b) expressing the first protein that is encoded by the nucleicacid; c) extracting cell lysate from the bacterial cell; d) a series ofpurifying steps comprising, in no particular order, removing theendogenous DNAK protein or DNAK protein homolog through a method thattargets the second tag; and e) purifying the first protein encoded bythe first nucleic acid by a method that binds to the first tag.
 9. Themethod of claim 8 wherein said first tag and second tag are epitopetags.
 10. The method of claim 8 wherein the purifying step utilizes asolid phase support or liquid phase support isolation procedure.
 11. Themethod of claim 8 wherein the purifying step may be repeated at leastonce.
 12. The method of claim 8 wherein other purifying steps areemployed that target other contaminants within the cell lysate.
 13. Themethod of claim 8 wherein removing the endogenous DNAK protein or DNAKprotein homolog also removes another protein that is associated with theendogenous DNAK protein or DNAK protein homolog.